This invention relates to background reduction devices and methods and more particularly to background reduction in confocal fluorescence detection systems.
Biological molecules are often tagged with fluorescent marker molecules for identification in analytical instruments in which the biological molecules are supported on a surface. The fluorescent signals originating from the surface (e.g., a glass substrate with fluorescently labeled molecules bound to it) can be weak so that the detection limit is often set by background radiation that comes from sources that lie outside of an intended detection plane. The contribution of such sources to the detected signal can be reduced substantially by using a confocal system as described extensively in the literature. See, for example, Handhook of Biological Confocal Microscopy, James Pawley, Ed. As is well known, confocal systems as used in scanners and microscopes reduce signal from out-of-plane sources. However, confocal systems have a normalized detection probability that goes to zero only asymptotically as a function of increasing distance from the intended plane of detection.
As an example, in the case of scanning a chip in a cartridge such as the Affymetrix GeneChip, non-negligible signals can be caused by fluorescent deposits on the front of the glass of the chip as well as by the illuminating laser beam hitting the back wall of the liquid cell in the chip cartridge. Confocality alone may not provide sufficient attenuation of such unwanted signals. One approach to reducing out-of-plane signals (such as from a back wall) when the confocal depth discrimination is insufficient is off-axis detection as used in a Hewlett-Packard G2500A system. However, off-axis detection is difficult to modify for large numerical aperture, i.e., high light collection efficiency. The present invention aims at specifically reducing unwanted background contributions for which confocality alone may not provide adequate suppression.
In one aspect, the background reduction system according to the invention is a confocal optical system for illuminating an object at an intended detection plane and to focus light emanating from the object onto a pinhole. An opaque obstacle is disposed within the confocal optical system, the obstacle selected to suppress light originating outside the intended detection plane from passing through the pinhole. The obstacle size and location are selected to achieve a desired level of suppression. The obstacle may be an opaque bulk obstacle or an opaque thin film supported, for example, on a transparent substrate. A bulk obstacle may also be supported by a beam or wire.
In another aspect, the invention is a method for determining obstacle size in a confocal optical system including a pinhole for light suppression outside of an object plane including calculating the diameter of a pinhole image in the object plane. The diameter of the percentage of encircled energy of an illuminating light source to be blocked is calculated in a plane of origin of the radiation to be blocked. Rays are traced back through edges of the pinhole image circle and the circle of encircled energy to a plane in which the obstacle is to be placed. The smallest circle that encircles loci of the rays in the obstacle plane is determined and the diameter of the smallest circle serves as the diameter of the obstacle. The center of this smallest circle serves as the center of the obstacle.
A confocal detection system is thus improved by placing an obstacle between the object plane and the confocal system pinhole. The size of the obstacle determines the degree of suppression of signals outside the intended detection plane. The bigger the obstacle, the higher the suppression will generally be. However, as the obstacle becomes bigger, it also reduces the transmission of signals from the intended detection plane. There is thus a trade-off to be made between background suppression and signal loss. The diameter of the obstacle may be increased to allow for alignment tolerances.